Francisella glycoconjugate vaccines

ABSTRACT

The disclosure relates to a glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen.

FIELD OF THE INVENTION

The disclosure relates to a vaccine comprising a glycoconjugateantigenic polypeptides conferring protection against Francisellatularensis infections and a method of glycosylating a polypeptideantigen.

BACKGROUND TO THE INVENTION

Subunit vaccines, based on proteins or polysaccharides displayed on thesurface of the pathogen are often preferred over inactivated orattenuated pathogens as they are known to cause fewer side effects.However, the development of a subunit vaccine is laborious requiring theidentification and isolation of protective antigens from the pathogenicorganism, and moreover subunit vaccines often invoke an immune responsewith low antibody titre, antibodies are short half-life and show lowaffinity for a specific antigen. It is known that the immunogenicity ofpolysaccharide antigens can be enhanced by conjugation to a proteincarrier. Currently licensed human glycoconjugate vaccines include thoseagainst Haemophilus influenzae, Neisserria meningitidis andStreptococcus pneumonia. These glycoconjugate vaccines use bacterialpolysaccharides that are chemically bound to carrier proteins. However,although these vaccines are effective, their production requires boththe purification of polysaccharide glycan from the native pathogen andthe chemical coupling of the sugar to a suitable protein carrier whichcan lead to low yields and variation between batches of conjugatesmaking this process is highly costly, inefficient and time consuming.

Several pathogenic bacteria have been identified forming glycoproteinssuch as for example the Gram negative pathogenic bacterium Campylobacterjejuni which harbours a protein glycosylation locus comprising pglA-pglGknown to be involved in the glycosylation of over 30 glycoproteins. Partof the gene cluster is PglB, an oligosaccharyltransferase catalysing thetransfer of glycans on to a wide range of different non-species relatedprotein acceptors indicating broad substrate specificity. Production ofglycoconjugate vaccines comprising a protein carrier and an antigenicpolysaccharide O-antigen from Shigella, E. coli and Pseudomonasaeruginosa using the oligosaccharyltransferase PglB in a bacterialsystem are disclosed in WO2009/104074.

Tularemia, also known as lemming or rabbit fever, is common in wildrodents and can be passed on to humans by contact with infected animaltissues, ticks or biting flies, or by inhalation of the infectiousorganism. Tularemia, a highly infectious disease with mortality rates upto 30% is found in North America, parts of Europe and Asia and is causedby the Gram-negative coccobacillus Francisella tularensis. Thedevelopment of vaccines protective against Francisella tularensisinfections are greatly desired as there are no effective treatmentmethods available. Lipopolysaccharide (LPS) comprising an O-antigen fromF. tularensis has shown protective effects in a murine infection model.However, the development of glycoprotein vaccines protecting againsthighly infectious pathogens are often associated with high safetyconcerns. Our pending application U.S. Ser. No. 14/655,210[WO2014/114926] the content of which is incorporated by reference in itsentirety, discloses vaccine compositions comprising an antigenicpolysaccharide isolated from Francisella tularensis which is linked tovarious carrier polypeptides. The glycoconjugates are produced by abacterial protein glycan coupling technology (PGCT) that allows the safeproduction of protective vaccines from the highly virulent wild-typestrains of F. tularensis holarctica and subsequent purification. Thesevaccines provide significant protection against subsequent challengeswhen compared to other LPS based vaccine treatments.

The disclosure relates to vaccines comprising alternative carriers withone or more glycosylation motifs for glycosylation. The glycosylatedcarriers show effective protection against F. tularensis infections. Inaddition we disclose whole cell glycosylation systems that use modifiedbacterial cells for the glycosylation of the carriers with FrancisellaO-antigen, in particular bacterial cells that have a non-functional WecA gene or a non-functional Wec A protein and the effect of expressinggenes involved in synthesizing Francisella O-antigen glycoconjugates ina Wec A⁺ and Wec N genetic background. Wec A is an integral membraneprotein that catalyses the transfer of N-acetylglucosamine(GlcNAC)-1-phosphate to undecaprenyl phosphate (Und-P) to formUnd-P-P-GlNAc [Lehrer et al (2007) Journal of Bacteriology 189:2617-2628].

STATEMENT OF INVENTION

According to an aspect of the invention there is provided a vaccinecomposition comprising: a carrier polypeptide comprising one or moreT-cell dependent epitopes and one or more amino acid sequences having atleast one amino acid motif comprising the amino acid sequenceD/E-X-N-X-S/T wherein X is any amino acid except proline and crosslinkedto said carrier polypeptide an antigenic polysaccharide wherein thepolysaccharide is isolated from Francisella and is an O-antigen.

According to an aspect of the invention there is provided an immunogeniccomposition comprising: a carrier polypeptide comprising one or moreT-cell dependent epitopes and one or more amino acid sequences havingthe amino acid motif D/E-X-N-X-S/T wherein X is any amino acid exceptproline and crosslinked to said carrier polypeptide is an antigenicpolysaccharide wherein the polysaccharide is isolated from Francisellaand is an O-antigen.

The term “carrier” is construed in the following manner. A carrier is animmunogenic molecule which, when bound to a second molecule, augmentsimmune responses to the latter. Some antigens are not intrinsicallyimmunogenic yet may be capable of generating antibody responses whenassociated with a foreign protein molecule such as keyhole-limpethaemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes butno T cell epitopes. The protein moiety of such a conjugate (the“carrier” protein) provides T-cell epitopes which stimulate helperT-cells that in turn stimulate antigen-specific B-cells to differentiateinto plasma cells and produce antibody against the antigen. Examples ofglycosylation motifs are set out in SEQ ID NO: SEQ ID NO: 18, SEQ ID NO19, SEQ ID NO: 20 and SEQ ID NO: 21 and are merely illustrative.

In a preferred embodiment of invention said O-antigen comprises:

2-acetamido-2-deoxy-O-D-galacturonamide, 4,6-dideoxy-4-formamido-D-glucose and/or 2-acetamido-2,6-dideoxy-O-D-glucose and/or N-acetyl glucosamine.

In a preferred embodiment of the invention said O-antigen comprises orconsists of:

4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNAcAN is 2-acetamido-2-deoxy-O-D-galacturonamide, Qui4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose.

In an alternative embodiment of the invention said O-antigen comprisesor consists of:

4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-GlcNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNacAN is 2-acetomido-2-deoxy-O-D-galacturonamide, Qui4NFm is4, 6-dideoxy-4-formamido-D-glucose and the reducing end group GlcNAc isN-acetyl glucosamine.

In a preferred embodiment of the invention said O-antigen is atetrasaccharide.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 1 or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 2 or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 3 or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 4 or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 5 or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 6 or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 7, or a polypeptide fragment comprising one ormore glycosylation motifs.

“Polymorphic sequence variant” is a nucleotide or amino acid sequencevariant that varies from a nucleotide or amino acid sequence and encodesor has an activity comparable or enhanced when compared to the subjectsequence. Typically, polymorphic sequences comprise nucleotide or aminoacid sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical over the full length sequence or part thereof.

“Polypeptide fragment” is a fragment of a full length polypeptide butcomprises at least one glycosylation motif and is at least 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids in length.

In an alternative embodiment of the invention said carrier polypeptidecomprises an amino acid sequence as set forth in SEQ ID NO: 1, 2, 3, 4,5, 6, or 7.

In a preferred embodiment of the invention said composition includes anadjuvant.

In a preferred embodiment of the invention said adjuvant is selectedfrom the group consisting of: cytokines selected from the groupconsisting of e.g. GMCSF, interferon gamma, interferon alpha, interferonbeta, interleukin 12, interleukin 23, interleukin 17, interleukin 2,interleukin 1, TGF, TNFα, and TNFβ.

In a further alternative embodiment of the invention said adjuvant is aTLR agonist such as CpG oligonucleotides, flagellin, monophosphoryllipid A, poly I:C and derivatives thereof.

In a preferred embodiment of the invention said adjuvant is a bacterialcell wall derivative such as muramyl dipeptide (MDP) and/or trehalosedycorynemycolate (TDM).

In a preferred embodiment of the invention said adjuvant is an aluminiumbased adjuvant suitable for use in a human subject.

In a preferred embodiment of the invention said adjuvant is selectedfrom the group of consisting of: aluminium hydroxide, aluminiumphosphate or calcium phosphate.

Adjuvants (immune potentiators or immunomodulators) have been used fordecades to improve the immune response to vaccine antigens. Theincorporation of adjuvants into vaccine formulations is aimed atenhancing, accelerating and prolonging the specific immune response tovaccine antigens. Advantages of adjuvants include the enhancement of theimmunogenicity of weaker antigens, the reduction of the antigen amountneeded for a successful immunisation, the reduction of the frequency ofbooster immunisations needed and an improved immune response in elderlyand immunocompromised vaccinees. Selectively, adjuvants can also beemployed to optimise a desired immune response, e.g. with respect toimmunoglobulin classes and induction of cytotoxic or helper T lymphocyteresponses. In addition, certain adjuvants can be used to promoteantibody responses at mucosal surfaces. Aluminium hydroxide andaluminium or calcium phosphate has been used routinely in humanvaccines. More recently, antigens incorporated into IRIV's(immunostimulating reconstituted influenza virosomes) and vaccinescontaining the emulsion-based adjuvant MF59 have been licensed incountries. Adjuvants can be classified according to their source,mechanism of action and physical or chemical properties. The mostcommonly described adjuvant classes are gel-type, microbial,oil-emulsion and emulsifier-based, particulate, synthetic and cytokines.More than one adjuvant may be present in the final vaccine product. Theymay be combined together with a single antigen or all antigens presentin the vaccine, or each adjuvant may be combined with one particularantigen. The origin and nature of the adjuvants currently being used ordeveloped is highly diverse. For example, aluminium based adjuvantsconsist of simple inorganic compounds, PLG is a polymeric carbohydrate,virosomes can be derived from disparate viral particles, MDP is derivedfrom bacterial cell walls; saponins are of plant origin, squalene isderived from shark liver and recombinant endogenous immunomodulators arederived from recombinant bacterial, yeast or mammalian cells. There areseveral adjuvants licensed for veterinary vaccines, such as mineral oilemulsions that are too reactive for human use. Similarly, completeFreund's adjuvant, although being one of the most powerful adjuvantsknown, is not suitable for human use.

In a preferred embodiment of the invention said composition includes atleast one additional anti-bacterial agent.

In a preferred embodiment of the invention said additionalanti-bacterial agent is a different antigenic molecule.

In a preferred embodiment of the invention said composition is amultivalent antigenic composition.

In an alternative preferred embodiment of the invention said additionalanti-bacterial agent is an antibiotic.

In a preferred embodiment of the invention said vaccine or immunogeniccomposition is formulated to be delivered as an aerosol.

According to a further aspect of the invention there is provided avaccine composition according to the invention for use in the preventionor treatment of a Francisella infection in a subject.

Preferably said infection is caused by Francisella tularensis.

In a preferred embodiment of the invention said vaccine composition isformulated to be delivered as an aerosol.

In a preferred embodiment of the invention said subject is an immunecompromised subject.

According to a further aspect of the invention there is provided amethod to treat a Francisella infection in a subject comprisingadministering an effective amount of a vaccine or immunogeniccomposition according to the invention.

Preferably said infection is caused by Francisella tularensis.

In a preferred method of the invention said vaccine composition isformulated to be delivered as an aerosol.

In a preferred method of the invention said subject is an immunecompromised subject.

According to an aspect of the invention there is provided an antigenicpolypeptide comprising: a carrier polypeptide comprising one or moreT-cell dependent epitopes and one or more amino acid sequences having atleast one amino acid motif comprising the amino acid sequenceD/E-X-N-X-S/T wherein X is any amino acid except proline and linked tosaid carrier polypeptide is an antigenic polysaccharide wherein thepolysaccharide is isolated from Francisella and is an O-antigen.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 1, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 2, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 3, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 4, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 5, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 6, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 7, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptidecomprises an amino acid sequence as set forth in of SEQ ID NO: 1, 2, 3,4, 5, 6, or 7.

In a preferred embodiment of invention said O-antigen comprises:

2-acetamido-2-deoxy-O-D-galacturonamide, 4,6-dideoxy-4-formamido-D-glucose and/or 2-acetamido-2,6-dideoxy-O-D-glucose and/or N-acetyl glucosamine.

In a preferred embodiment of the invention said O-antigen comprises orconsists of:4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNAcAN is 2-acetamido-2-deoxy-O-D-galacturonamide, Qui4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose.

In an alternative embodiment of the invention said O-antigen comprisesor consists of:4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-GlcNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNacAN is 2-acetomido-2-deoxy-O-D-galact-uronamide, Qui4NFm is4, 6-dideoxy-4-formamido-D-glucose and the reducing end group GlcNAc isN-acetyl glucosamine.

According to a further aspect of the invention there is provided abacterial cell wherein said cell is genetically modified to include:

-   -   i) a nucleic acid molecule comprising the nucleotide sequence of        the Francisella O-antigen biosynthetic polysaccharide locus [SEQ        ID NO: 8];    -   ii) a nucleic acid molecule comprising a nucleotide sequence of        an oligosaccharyltransferase; and/or    -   iii) a nucleic acid molecule comprising a nucleotide sequence of        carrier polypeptide wherein the a carrier polypeptide comprising        one or more T-cell dependent epitopes and one or more amino acid        sequences having at least one amino acid motif comprising the        amino acid sequence D/E-X-N-X-S/T wherein X is any amino acid        except proline, wherein said bacterial cell is adapted for        expression of each nucleic acid molecule and synthesizes an        antigenic polypeptide according to the invention.

In a preferred embodiment said nucleic acid molecule encoding anoligosaccharyltransferase comprises a nucleotide sequence set forth inSEQ ID NO: 9, 23, 25 or 26, or a polymorphic sequence variant thereof,wherein said variant comprises a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 9, 23, 25 or 26 and wherein saidnucleic acid molecule encodes an oligosaccharyltransferase.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 1, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 2, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 3, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 4, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 5, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 6, or a polypeptide fragment comprising one ormore glycosylation motifs.

In a preferred embodiment of the invention said carrier polypeptide, orpolymorphic sequence variant, comprises an amino acid sequence as setforth in of SEQ ID NO: 7, or a polypeptide fragment comprising one ormore glycosylation motifs.

In an alternative embodiment of the invention said carrier polypeptidecomprises an amino acid sequence as set forth in of SEQ ID NO: 1, 2, 3,4, 5, 6 or 7.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 10, or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 10.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 11, or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 11.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 13 or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 13.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 14 or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 14.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 15 or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 15.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 16 or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 16.

In a preferred embodiment of the invention said carrier polypeptide isencoded by a nucleic acid molecule comprising a nucleotide sequence asset forth in SEQ ID NO: 29 or a nucleic acid molecule the complementarystrand of which hybridizes under stringent hybridization conditions tothe sequence set forth in SEQ ID NO: 29.

In a preferred embodiment said bacterial cell expresses aglycosyltransferase encoded by a nucleic acid molecule comprising thenucleic acid sequence as set forth in SEQ ID NO 17, or a sequencevariant thereof wherein said variant comprises a nucleic acid moleculethe complementary strand of which hybridizes under stringenthybridization conditions to the sequence set forth in SEQ ID NO: 17 andwherein said nucleic acid molecule encodes an glycosyltransferase;

In a preferred embodiment said glycosyltransferase encoded by a nucleicacid molecule comprising the nucleic acid sequence set forth in SEQ IDNO 17 is modified wherein said modification is the addition,substitution or deletion of at least one nucleic acid base wherein saidmodified nucleic acid sequence encodes a glycosyltransferase polypeptidewhich has reduced or undetectable enzyme activity when compared to anunmodified glycosyltransferase polypeptide.

In a preferred embodiment of the invention said bacterial cell isdeleted for all or part of the nucleic acid sequence set forth in SEQ IDNO: 17 to provide a non-functional glycosyltransferase polypeptide.

Hybridization of a nucleic acid molecule occurs when two complementarynucleic acid molecules undergo an amount of hydrogen bonding to eachother. The stringency of hybridization can vary according to theenvironmental conditions surrounding the nucleic acids, the nature ofthe hybridization method, and the composition and length of the nucleicacid molecules used. Calculations regarding hybridization conditionsrequired for attaining particular degrees of stringency are discussed inSambrook et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes Part I, Chapter 2(Elsevier, New York, 1993). The T, is the temperature at which 50% of agiven strand of a nucleic acid molecule is hybridized to itscomplementary strand.

The following is an exemplary set of hybridization conditions and is notlimiting.

Very High Stringency (Allows Sequences that Share at Least 90% Identityto Hybridize)

-   -   a) Hybridization: 5×SSC at 65° C. for 16 hours    -   b) Wash twice: 2×SSC at room temperature (RT) for 15 minutes        each    -   c) Wash twice: 0.5×SSC at 65° C. for 20 minutes each

High Stringency (Allows Sequences that Share at Least 80% Identity toHybridize)

-   -   a) Hybridization: 5×-6×SSC at 65° C.-70° C. for 16-20 hours    -   b) Wash twice: 2×SSC at RT for 5-20 minutes each    -   c) Wash twice: 1×SSC at 55° C.-70° C. for 30 minutes each

Low Stringency (Allows Sequences that Share at Least 50% Identity toHybridize)

-   -   i) Hybridization: 6×SSC at RT to 55° C. for 16-20 hours    -   ii) Wash at least twice: 2×-3×SSC at RT to 55° C. for 20-30        minutes each.

A variant oligosaccharyltransferase polypeptide as herein disclosed maydiffer in amino acid sequence by one or more substitutions, additions,deletions, truncations that may be present in any combination. Amongpreferred variants are those that vary from a reference polypeptide byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid by another amino acid of likecharacteristics. The following non-limiting list of amino acids areconsidered conservative replacements (similar): a) alanine, serine, andthreonine; b) glutamic acid and aspartic acid; c) asparagine andglutamine d) arginine and lysine; e) isoleucine, leucine, methionine andvaline and f) phenylalanine, tyrosine and tryptophan. Most highlypreferred are variants that retain or enhance the same biologicalfunction and activity as the reference polypeptide from which it varies.

In one embodiment, the variant polypeptides have at least 50% or 55%identity, more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% identity, or at least 99% identity with the full length amino acidsequence illustrated herein.

In a preferred embodiment of the invention at least theoligosaccharyltransferase of ii) above is integrated into the bacterialgenome to provide a stably transfected and expressingoligosaccharyltransferase.

In a further preferred embodiment of the invention one or more nucleicacid molecules encoding carrier polypeptides are also integrated intothe bacterial genome.

According to a further aspect of the invention there is provided abacterial cell culture comprising a genetically modified bacterial cellaccording to the invention.

According to a further aspect of the invention there is provided aprocess for the production of one or more glycoconjugates comprising:

-   -   i) providing a bacterial cell culture according to the        invention;    -   ii) providing cell culture conditions; and    -   iii) isolating one or more glyconjugates from the bacterial cell        or cell culture medium.

According to a further aspect of the invention there is provided a cellculture vessel comprising a bacterial cell culture according to theinvention.

In a preferred embodiment of the invention said cell culture vessel is afermentor.

Bacterial cultures used in the process according to the invention aregrown or cultured in the manner with which the skilled worker isfamiliar, depending on the host organism. As a rule, bacteria are grownin a liquid medium comprising a carbon source, usually in the form ofsugars, a nitrogen source, usually in the form of organic nitrogensources such as yeast extract or salts such as ammonium sulfate, traceelements such as salts of iron, manganese and magnesium and, ifappropriate, vitamins, at temperatures of between 0° C. and 100° C.,preferably between 10° C. and 60° C., while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to sayregulated during the culturing period, or not. The cultures can be grownbatchwise, semi-batchwise or continuously. Nutrients can be provided atthe beginning of the fermentation or fed in semi-continuously orcontinuously. The products produced can be isolated from the bacteria asdescribed above by processes known to the skilled worker, for example byextraction, distillation, crystallization, if appropriate precipitationwith salt, and/or chromatography. In this process, the pH value isadvantageously kept between pH 4 and 12, preferably between pH 6 and 9,especially preferably between pH 7 and 8.

An overview of known cultivation methods can be found in the textbookBioprocess technology 1. Introduction to Bioprocess technology (GustavFischer Verlag, Stuttgart, 1991) or in the textbook by Storhas(Bioreaktoren and periphere Einrichtungen [Bioreactors and peripheralequipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must suitably meet the requirements of thebacterial strains in question. Descriptions of culture media for variousbacteria can be found in the textbook “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981).

As described above, these media which can be employed in accordance withthe invention usually comprise one or more carbon sources, nitrogensources, inorganic salts, vitamins and/or trace elements.

Preferred carbon sources are sugars, such as mono-, di- orpolysaccharides. Examples of carbon sources are glucose, fructose,mannose, galactose, ribose, sorbose, ribulose, lactose, maltose,sucrose, raffinose, starch or cellulose. Sugars can also be added to themedia via complex compounds such as molasses or other by-products fromsugar refining. The addition of mixtures of a variety of carbon sourcesmay also be advantageous. Other possible carbon sources are oils andfats such as, for example, soya oil, sunflower oil, peanut oil and/orcoconut fat, fatty acids such as, for example, palmitic acid, stearicacid and/or linoleic acid, alcohols and/or polyalcohols such as, forexample, glycerol, methanol and/or ethanol, and/or organic acids suchas, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials comprising these compounds. Examples of nitrogen sourcescomprise ammonia in liquid or gaseous form or ammonium salts such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate or ammonium nitrate, nitrates, urea, amino acids or complexnitrogen sources such as cornsteep liquor, soya meal, soya protein,yeast extract, meat extract and others. The nitrogen sources can be usedindividually or as a mixture.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphorus and sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or elseorganic sulfur compounds such as mercaptans and thiols may be used assources of sulfur for the production of sulfur-containing finechemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used assources of phosphorus.

Chelating agents may be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate and organic acidssuch as citric acid.

The fermentation media used according to the invention for culturingbacteria usually also comprise other growth factors such as vitamins orgrowth promoters, which include, for example, biotin, riboflavin,thiamine, folic acid, nicotinic acid, panthothenate and pyridoxine.Growth factors and salts are frequently derived from complex mediacomponents such as yeast extract, molasses, cornsteep liquor and thelike. It is moreover possible to add suitable precursors to the culturemedium. The exact composition of the media compounds heavily depends onthe particular experiment and is decided upon individually for eachspecific case. Information on the optimization of media can be found inthe textbook “Applied Microbiol. Physiology, A Practical Approach”(Editors P. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN0 19 963577 3). Growth media can also be obtained from commercialsuppliers, for example Standard 1 (Merck) or BHI (brain heart infusion,DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 barand 121° C.) or by filter sterilization. The components may besterilized either together or, if required, separately. All mediacomponents may be present at the start of the cultivation or addedcontinuously or batchwise, as desired.

The culture temperature is normally between 15° C. and 45° C.,preferably at from 25° C. to 40° C., and may be kept constant or may bealtered during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for cultivation canbe controlled during cultivation by adding basic compounds such assodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia oracidic compounds such as phosphoric acid or sulfuric acid. Foaming canbe controlled by employing antifoams such as, for example, fatty acidpolyglycol esters. To maintain the stability of plasmids it is possibleto add to the medium suitable substances having a selective effect, forexample antibiotics. Aerobic conditions are maintained by introducingoxygen or oxygen-containing gas mixtures such as, for example, ambientair into the culture. The temperature of the culture is normally 20° C.to 45° C. and preferably 25° C. to 40° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyachieved within 10 to 160 hours.

The fermentation broth can then be processed further. The biomass may,according to requirement, be removed completely or partially from thefermentation broth by separation methods such as, for example,centrifugation, filtration, decanting or a combination of these methodsor be left completely in said broth. It is advantageous to process thebiomass after its separation.

However, the fermentation broth can also be thickened or concentratedwithout separating the cells, using known methods such as, for example,with the aid of a rotary evaporator, thin-film evaporator, falling-filmevaporator, by reverse osmosis or by nanofiltration. Finally, thisconcentrated fermentation broth can be processed to obtain the fattyacids present therein.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, means “including but not limited to”, andis not intended to (and does not) exclude other moieties, additives,components, integers or steps. “Consisting essentially” means having theessential integers but including integers which do not materially affectthe function of the essential integers.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

An embodiment of the invention will now be described by example only andwith reference to the following figures:

FIG. 1: Principles of Protein Glycan Coupling Technology in E. coli. AnE. coli cell is transformed with three plasmids to generate the clonedglycoconjugate protein (GP). The plasmids encode theoligosaccharyltransferase PglB, the biosynthetic polysaccharide locusand the carrier protein. The polysaccharide is synthesised on anundecaprenol pyrophosphate lipid anchor (●) within the cytoplasm, thisis transferred to the periplasmic compartment where PglB recognises thelipid link reducing end sugar and transfers the polysaccharide en bloconto an acceptor sequon (D/E-X-N-X-S/T) on the carrier protein toproduce the glycoconjugate protein (GP). IM, inner membrane; OM, outermembrane;

FIG. 2: Western blot of glycoconjugates DnaK-FT and IglC indicate theseF. tularensis proteins are glycosylated by C. jejuni PglB with the F.tularensis O-antigen. These are produced in large quantity indicatingthe suitability of DnaK and IglC as protective vaccine candidates. M:protein marker, DnaK and IglC glycoconjugates were prepared in largescale 2 L batch culture and purified. Western blot indicates protein(green) and F. tularensis O-antigen glycan (red) both present insamples;

FIG. 3: amino acid sequence of carrier protein DnaK (Francisellatularensis) (SEQ ID NO 2);

FIG. 4: amino acid sequence of carrier protein IglC (Francisellatularensis) (SEQ ID NO 1);

FIG. 5: nucleotide sequence encoding the Francisella O-antigenbiosynthetic polysaccharide (SEQ ID NO 8);

FIG. 6: nucleotide sequence encoding the oligosaccharyltransferase PglB(C. jejuni) (SEQ ID NO 9);

FIG. 7: Pgl B amino acid sequence (C. jejuni) (SEQ ID NO 22);

FIG. 8: nucleotide sequence of carrier protein DnaK (Francisellatularensis) (SEQ ID NO 11);

FIG. 9: nucleotide acid sequence of carrier protein IglC (Francisellatularensis) (SEQ ID NO 10);

FIG. 10: amino acid sequence of carrier protein ExoA (Pseudomonasaeruginosa) (SEQ ID NO 3);

FIG. 11: amino acid sequence of carrier protein TUL4 (Francisellatularensis) (SEQ ID NO 4);

FIG. 12: amino acid sequence of carrier protein FTT1713c (Francisellatularensis) (SEQ ID NO 5);

FIG. 13: amino acid sequence of carrier protein FTT1695 (Francisellatularensis) (SEQ ID NO 6);

FIG. 14: amino acid sequence of carrier protein FTT1696 (Francisellatularensis) (SEQ ID NO 7);

FIG. 15: Pgl B amino acid sequence (C. jejuni) (SEQ ID NO 22);

FIG. 16: nucleotide sequence encoding the oligosaccharyltransferase PglB(Campylobacter sputorum) (SEQ ID NO 23);

FIG. 17: amino acid sequence (full length) of PglB (Campylobactersputorum) (SEQ ID NO 24).

FIG. 18: P. aeruginosa ExoA with additional glycosylation sequons isheavily glycosylated by F. tularensis O-antigen by C. jejuni PglB in E.coli CLM24. Two-colour Western blots were used to simultaneously detectthe degree of glycosylation of ExoA using monoclonal mouse mAb FB011 (a)(red) and rabbit anti-6× His (b) (green). The two IR secondary antibodychannels when overlaid (IR 800/680) result in images with overall yellowcolour indicating conjugation (c). Marker, PageRuler Plus Prestainedprotein ladder (Life technologies); Lane 1, pGVXN150 only; Lane 2,pGVXN150 ExoA glycosylated with the F. tularensis O-antigen (sameconstruct from Cuccui et al., 2013); Lane 3, ExoA heavily glycosylatedwith the F. tularensis O-antigen due to additional terminalglycosylation sequons;

FIG. 19: Survival (A) and disease of rats following aerosol challengewith a range of doses of F. tularensis Schu S4. Groups of 5 Fischer 344rats were challenged via the aerosol route and monitored daily formortality. Indicated challenge doses represent calculated dose fromsampling of aerosol during challenge. Signs of disease in these animalswere monitored twice daily Average cumulative signs for each group ispresented for animals which had not succumbed to disease (B). Weight wasmonitored daily. Average weight change for each group is presented foranimals which had not succumbed to disease (C);

FIG. 20: Effect of rat white blood cell counts during vaccination withFt LVS Imaging flow cytometry was used to assess the effect of LVSvaccination on the rat immune response. Whole rat blood was labelledwith CD45, CD3 and counter stained with DAPI for nuclear visualisation.Quantification of lymphocytes, monocytes/macrophages and neutrophils wasachieved using CD45 vs side scatter (SSC) (A). Cell we gated as followsSSC high CD45 low for neutrophils, SSC low CD45 high for lymphocytes andSSC medium CD45 high for monocytes/macrophages (B). Data (C) shows the %gated of each cell type and each rat over the 21 day time course. Datashows a decrease in lymphocytes at day with returning to control levelsat day 7 and 21. It also shows increased in neutrophils at day returningto normal level at days 7 and 21. A significant increase inmonocytes/macrophages is only observed in the LVS 10⁷ group at day 3again this returns to normal levels by day 7 and 21.

BF=Brightfield

SSC=Side Scatter

Composite=CD45, CD3 and nucleus

*=p<0.01 by two-way ANOVA and Dunnett's post tests

Images are representative of populations gated and fluorescence markersare optimised for visual impact with IDEAS®;

FIG. 21: Antigen stimulated IFNγ response in LVS infected rats.Splenocytes isolated from rats 21 days following infection with 10⁵ CFULVS (grey bars), 10⁷ CFU LVS (black bars) or the PBS controls (hashedbars) were cultured in the presence of LVS sonicate, F. tularensissonicate, Con-A or medium and then expression of IFNγ in 72 hour culturesupernatants measured by ELISA. IFNγ responses (ρg/ml) are presented asmean response for each group (n=5)±SEM;

FIG. 22: Measurement of antigen stimulated intracellular expression ofIFNγ and IL17. Splenocytes isolated from LVS infected or PBS controlrats 21 days-post infection were cultured in the presence of LVSsonicate, F. tularensis sonicate, Con-A or medium and then intracellularco-expression of IFNγ and/or IL17 was determined by flow cytometry. Thepercentage of antigen stimulated CD4⁺ (panels A-C) and CD8⁺ (panels D-F)T-cells expressing either IFN and/or IL17 is presented for each of the 3treatment groups and for each of the antigens (medium control responsesubtracted). These data are presented by stacked bar graphs representingmean response for each group (n=5)±SEM;

FIG. 23: Antigen stimulated IFNγ response in glyco-conjugate vaccinatedrats. Splenocytes isolated from rats immunised with glyco-conjugatevaccine administered i.p. (light grey bars) or s.c. (dark grey bars),and from rats infected with LVS (black bars) or the PBS controls (hashedbars) were cultured in the presence of LVS sonicate, F. tularensissonicate, Con-A or medium. The expression of IFNγ in 72 hour culturesupernatants was measured by ELISA. Responses were measured 6 weeks postvaccination for glyco-conjugate and PBS groups and 3 weeks postinfection for the LVS group. IFNγ responses (ρg/ml) are presented asmean response for each group (n=5 except for LVS group where n=4)±SEM;

FIG. 24: Protection of Fischer 344 rats against aerosol delivered SchuS4 with LVS and glyco-conjugate Groups of five Fischer 344 rats werevaccinated three times, two weeks apart with 10 μg glyco conjugate inMF59 or MF59 alone via the s.c. or i.p. route, or 5.38×10⁷ LVS. 5 weeksafter final vaccination, rats were challenged with 5.48×10² Schu S4 viathe aerosol route and monitored twice daily for mortality (A). Signs ofdisease were recorded twice daily and average cumulative signs forsurviving rats in each group of 5 presented (B). Rats were weighed oncedaily. Presented data is average weight change of surviving rats ingroups of 5 (C). Each group is presented with its apposite control: LVSs.c. and PBS s.c. (C.1), G-tExoA+MF59 s.c. and MF59 s.c. (C.2),Gt-ExoA+MF59 i.p and MF59 i.p. (C.3). Significance in divergence ofweight change between groups is denoted as * p<0.05, ** p<0.005 and ***p<0.0005 for each day; and

FIG. 25: Example gating strategy used to measure intracellularexpression of IFNγ and IL17. Splenocytes were stimulated with eitherantigen (LVS and F. tularensis sonicates), mitogen (Con-A) or mediumcontrol prior to surface staining with rat CD3, CD4 and CD8 antibodiesand then intracellular staining for IFNγ and IL17. Histograms wereinitially gated in accordance with the following hierarchy, singletcells (histogram A), live cells (histogram B) and lymphocytes (histogramC) prior to gating for co-expression for CD3⁺CD4⁺ and CD3⁺CD8⁺(histogram D and E respectively). The example expression of IFNγ andIL17 from the CD4⁺ and CD8⁺ T-cell populations derived from splenocytesisolated from a rat infected with 10⁷ CFU LVS and then stimulated withF. tularensis sonicate antigen is presented in histograms F and G. Theequivalent response to the medium control stimulated splenocytes isshown in the corresponding inset histograms H and I to demonstrate theantigen-specific nature of the IFNγ⁺ and IL17⁺ response presented.

TABLE 1 Summary of Sequence Information SEQ ID NO Gene/protein 1 IglCprotein 2 DNAk protein 3 Exo A protein 4 Tul4 protein 5 FTT1713c protein6 FTT1695 protein 7 FTT1696 protein 8 Francisella O antigen locus DNA 9Campylobacter jejuni Pgl B DNA 10 IglC DNA 11 DNAk DNA 12 KnR 13 Tul4DNA 14 FTT1713c DNA 15 FTT1695 DNA 16 FTT1696 DNA 17 Wec A 18 DXNXS 19DXNXT 20 EXNXS 21 EXNXT 22 PglB C. jejuni 23 PglB1 C. sputorum 24 PglB1C. sputorum 25 PlgB2 C. sputorum 26 PlgB2 C. sputorum optimised 27 PglB2C. sputorum 28 KnF 29 ExoA DNA

TABLE 2 Strains and plasmids used Strain/plasmid Description Source E.coli Top10 F-mcrA Δ(mrr-hsdRMS- Invitrogen mcrBC) φ80lacZΔM15 ΔlacX74nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(Str^(R)) endA1 λ⁻E. coli DH5α F-φ80lacZΔM15 Invitrogen Δ(lacZYA-argF) U169 deoRrecA1endA1 hsdR17 (rk−, mk+), gal- phoAsupE44λ - thi-1 gyrA96 relA1 E. coliXL-1 endA1 gyrA96(nalr)thi-1 Stratagene relA1 lac gln V44 F′[::Tn10proAB+ laclq Δ (lacZ)M15] hsdR17 (r_(k) ⁻m_(k) ⁺) E. coli CLM24 rph-IIN(rrnD-rrnE) 1, 5 ΔwaaL F. tularensis subs. tularensis strain Type Astrain DSTL, Porton SchuS4 Down laboratories F. tularensis subs.holarctica strain HN63 Type B strain, isolated in Green, M., et al.,Norway from an infected Efficacy of the live Hare attenuated Francisellatularensis vaccine (LVS) in a murine model of disease. Vaccine, 2005.23(20): p. 2680-6 pGEM-T Easy TA cloning vector, amp^(r) Promega pGHVector construct GT-ExoA Celtek Bioscience, was synthesized in prior toLLC subcloning into pGVXN150 pLAFR1 Low copy expression Vanbleu E,vector, tet^(r) Marchal K, Vanderleyden J. Genetic and physical map ofthe pLAFR1 vector. DNA Seq. 2004 Jun; 15(3): 225-7. pGAB1 F. tularensisO antigen This study coding region inserted into MCS of pGEM-T easypGAB2 F. tularensis subs. This study tularensis strain SchuS4 O antigencoding region inserted into EcorI site of pLAFR. pGVXN114 Expressionplasmid for GlycoVaxyn CjPglB regulated from the Lac promoter in pEXT21.IPTG inducible, HA tag, Spec^(r). pGVXN115 Expression plasmid for C.jejuninon GlycoVaxyn functionalPglB due to a mutation at ₄₅₇WWDYGY₄₆₂ to₄₅₇WAAYGY_(462,) regulated from the Lac promoter in pEXT21. IPTGinducible, HA tag, Spec^(r). pGVXN150₂₆₀DNQNS₂₆₄ Expression plasmid forThis study Pseudomonas aeruginosa PA103 (DSM111/) Exotoxin A with thesignal peptide of the E. coli DsbA protein, two inserted bacterialN-glycosylation sites, AA at position 262 altered from N to Q and ahexahis tag at the C- terminus. Induction under control of an arabinoseinducible promoter, Amp^(r) pGVXN150₄₀₂DQQRT₄₀₆ Expression plasmid forThis study Pseudomonas aeruginosa PA103 (DSM111/) Exotoxin A with thesignal peptide of the E. coli DsbA protein, two inserted bacterialN-glycosylation sites, AA at position 404 altered from N to Q and ahexahis tag at the C- terminus. Induction under control of an arabinoseinducible promoter, Amp^(r) pGVXN150₂₆₀DNQNS₂₆₄/₄₀₂DQQRT₄₀₆ Expressionplasmid for This study Pseudomonas aeruginosa PA103 (DSM111/) Exotoxin Awith the signal peptide of the E. coli DsbA protein, two insertedbacterial N-glycosylation sites, AA at position 262 and 404 altered fromN to Q and a hexahis tag at the C-terminus. Induction under control ofan arabinose inducible promoter, Amp^(r) pGVXN150:GTExoA Expressionplasmid for P. aeruginosa This study PA103 Exotoxin A (ExoA) with thesignal peptide of the E. coli DsbA protein, two inserted bacterial N-glycosylation sites, two extra terminal glycosylation sites on the N andC-termini, and a hexa-His tag at the C- terminus. Induction undercontrol of an arabinose inducible promoter, Amp^(r) pACYCpgl pACYC184carrying the 5 CjPglB locus, Cm^(r) E. coli CedAPglB E. coli strainCLM24 with a This study chromosomally inserted IPTG inducible copy ofPglB pGVXN150 Expression plasmid for P. aeruginosa GlycoVaxyn, PA103ExoA Cuccui et al., 2013 with the signal peptide of the E. coli DsbAprotein, two inserted bacterial N- glycosylation sites, and a hexa-Histag at the C- terminus. Induction under control of an arabinoseinducible promoter, Amp^(r)

Materials and Methods

Bacterial Strains

Escherichia coli strains were grown in LB at 37° C., 180 rpm for smallscale tests, or 110 rpm for large scale vaccine production. Antibioticsand or supplements were used at the following concentrations;tetracycline 20 μg/ml, ampicillin 100 μg/ml, spectinomycin 80 μg/ml andchloramphenicol 30 μg/ml. The host strain for initial cloningexperiments was E. coli XL-1, subsequent strains used for glycoconjugateproduction were E. coli DH5a and CLM24 (Table 1). For efficacy studies,mice were challenged with F. tularensis subsp. holarctica strain HN63.The bacterium was cultured on blood cysteine glucose agar plates(supplemented with 10 ml of 10% (wt/vol) histidine per litre) at 37° C.for 18 hours.

For vaccination of rats with LVS, Lot 4, bacteria were inoculated ontoblood cysteine glucose agar (BCGA) and incubated at 37° C. for 48 h.Bacterial growth was recovered from the agar and re-suspended inphosphate buffered saline (PBS), and the OD₆₀₀ adjusted to 0.14. Thesuspension was serially diluted ten-fold to the desired concentrationfor immunisation.

For challenge studies, F. tularensis Schu S4 was inoculated onto BCGAand incubated at 37° C. for 24 h. Growth was recovered from agar,re-suspended in PBS and the OD₆₀₀ adjusted to 0.1. One ml of thissuspension was inoculated into 100 ml modified cysteine partialhydrolysate (MCPH) broth with 4% glucose and incubated with shaking at180 rpm, at 37° C. for 48 h. OD₆₀₀ of the culture was adjusted to 0.1 inPBS, and serially diluted to the desired concentration for aerosolchallenge.

To determine bacterial load in organs, organs were weighed, thenhomogenised through a 40 μm cell sieve, serially diluted in PBS, andplated onto BCGA.

Cloning, Sequencing and Expression of the F. tularensis O-Antigen CodingRegion

DNA was prepared from the F. tularensis subsp. tularensis strain SchuS4by phenol extraction as described by Karlsson et al. (2000). TheO-antigen coding region was amplified using the primers FTfragment2rev(5′-GGATCATTAATAGCTAAATGTAGTGCTG-3′; SEQ ID NO:30) and ° anti ftfwd(5′-TTTTGAATTCTACAGGCTGTCAATGGAGAATG-3′; SEQ ID NO:31) using thefollowing cycling conditions: 94° C., 15 sec, 55° C., 15 sec, 68° C., 20min; 35 cycles using Accuprime TaqHifi (Invitrogen U.K.). This wascloned into the TA cloning vector pGEM-T Easy to generate the vectorpGAB1. The plasmid pGAB1 was digested with EcoRI and the insert wassubcloned into the vector pLAFR to generate the construct pGAB2.

Immunofluorescence Imaging of E. coli Cells Carrying F. tularensis OAntigen Coding Region

Immunofluorescence was carried out as previously described [17] with themodification that the IgG2a mouse monoclonal antibody FB11 was used todetect F. tularensis O antigen (1 μl/ml in 5% (v/v) FCS/PBS).

Bacterial Strains and Plasmid Construction

Escherichia coli CLM24 (17) was used as the host strain for proteinexpression and glycoconjugate production. CLM24 (a ligase negativestrain) was stably transformed with the plasmid pGab2 (24), a constructcreated from insertion of the F. tularensis subspecies tularensis strainSchuS4 O-antigen into the low copy number expression plasmid pLAFR (25).pGab2 is tetracycline-selectable and constitutively expressed. Followingconfirmation of the expression of the F. tularensis O-antigen, theresulting strain was then transformed with the plasmid CLM24 contained aplasmid encoded C. jejuni PglB, pGVXN114, which expresses the C. jejuniglycotransferase pglB Finally, the resulting strain was transformed withthe plasmid pGVXN150:GT-ExoA, creating a three plasmid system forproduction of the glycoconjugate. The GT-ExoA construct was engineeredto expressed a modified version of P. aeruginosa Exotoxin A that wassynthesized by Celtek Bioscience, LLC in the vector pGH and closed intoa vector derived from pEC415 using the restriction enzymes NheI andEcoRI (NEB). The synthesized protein contains two internal modificationsthat allow glycosylation of the protein by PglB (24), as well ascontaining four N-glycosylation sequons at the N terminal and anadditional 4 at the C terminals glycotags. In addition, a hexa-histidinetag was added to the C-terminus of the protein to facilitatepurification and an and an E. coli DsbA signal peptide was added to theN-terminal sequences enabling Sec-dependent secretion to the periplasm.pGVXN150: GT-ExoA is ampicillin resistant and L-(+)—Arabinose inducible.The construct sequence was then confirmed using Sanger sequences withthe primers GTExoA NF (GCGCTGGCTGGTTTAGTTT, SEQ ID NO 32), GTExoA NR(CGCATTCGTTCCAGAGGT, SEQ ID NO 33), GTExoA CF (GACAAGGAACAGGCGATCAG, SEqID NO 34) and GTExoA CR (TGGTGATGATGGTGATGGTC, SEQ ID NO 35).

Culture and Glycoprotein Expression Conditions

For all experiments, E. coli CLM24 was cultured in Luria-Bertani (LB)broth (Fisher Scientific) supplemented with appropriate antibiotics inthe following concentrations: ampicillin 100 μg/mL, tetracycline 20μg/mL, and spectinomycin 80 μg/mL. The addition of manganese chloride atthe time of protein and PglB induction was at a final concentration of 4mM, and made up as a 1 M stock fresh prior to each experiment. Cultureswere incubated at 37° C. at 110 RPM for 16-20 hrs for large-scalepreparation. For three plasmid system glycoconjugate production, anovernight LB culture of E. coli CLM24 harbouring p114, pGvn150:GT-ExoAand pGab2 was sub-cultured in a 1:10 dilution of LB broth (FisherScientific) with antibiotics, and grown to mid log phase.pGVXN150:GT-ExoA was induced by addition of 0.2% L-(+)-Arabinose(Sigma), and C. jejuni PglB induced with 1 mM IPTG, followed byincubation for an initial 4 hours. Another addition of 0.4%L-(+)-Arabinose was then added and cultures were incubated overnight.

Production and Purification of Glycoconjugate Vaccine

E. coli CLM24 carrying the vectors pGAB2, pGVXN114 and pGVXN150 wasgrown for 16 h in 200 mL LB broth at 37° C., 180 rpm. This was used toinoculate 1.8 L of LB broth and further incubated at 110 r.p.m. 37° C.to an OD.600 nm reading of 0.4 to 0.6. L-(+)-arabinose was then added toa final concentration of 0.2% w/v and IPTG to a final concentration of 1mM to induce expression of ExoA and Cj PglB respectively; At this pointin time manganese chloride at a final concentration of 4 mM was alsoadded. Following 5 hours of incubation, 0.2% w/v L-(+)-arabinose wasadded again and the culture left to incubate overnight.

Cells were harvested by centrifugation at 5300 g for 30 min, andpelleted cells were then resuspended in an ice cold lysis solutioncomposed of 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0(adjusted with 5 M NaOH) supplemented with 1 mg/ml lysozyme and 1 μl/mlBenzonase nuclease (Novagen). Then, cells were subjected to five roundsof lysis using a pre-chilled Stansted High Pressure Cell Disruptor(Stansted Fluid Homogenizer) under ˜60,000 psi in continuous mode. Celldebris was removed by centrifugation at 10,000 r.p.m. for 60 m, thesupernatant was collected The resulting supernatant was kept on icewhile being loaded onto a GE Healthcare HIS trap HP 1 mL column. Then,the column was washed in buffer containing 50.0 mM NaH₂PO₄; 300 mMNaCl₂; 20 mM imidazole lysis while attached to an AKTA purifier.Material was eluted and collected in 1 mL fractions with an imidazolegradient of 30-500 mM elution buffer that also contained 20% v/vglycerol and 5 w/v glucose. The collected fractions were then visualisedby Western blot and the most glycosylated F. tularensis carrier proteinsconjugated to F. tularensis O-antigen were chosen for pooling and bufferexchange (using VivaSpin 2 (VivaProducts) into PBS 20 v/v glycerol,prior to quantification with a BCA Protein Assay Kit (PierceBiotechnology, USA). This generated a typical yield of 2-3.5 mg/ml ofglycoconjugate per 2 L of E. coli culture.

The same techniques were used for the generation of the ‘sham’ C. jejuniheptasaccharide ExoA glycoconjugate encoded by pACYCpgl [18].

Using the E. coli Chromosomally Inserted Strain CLM 24 CedAPglB:

Escherichia coli strain CLM24 with a chromosomally inserted copy of pglBwere grown in Luria-Bertani (LB) broth at 37° C., with shaking.Antibiotics were used at the following concentrations: tetracycline 20μg ml⁻¹ and ampicillin 100 μg ml⁻¹. Tetracycline was used to maintainthe plasmid pGAB2 coding for Francisella tularensis O antigen andampicillin was used to maintain the plasmid coding for the acceptorcarrier protein.

E. coli cells were grown for 16 h in 200 ml LB broth at 37° C., withshaking. This was used to inoculate 1.8 l of LB broth and furtherincubated with shaking at 37° C. until an OD₆₀₀ reading of 0.4-0.6 wasreached. At this point L-(+)-arabinose was added to a finalconcentration of 0.2% w/v and IPTG to a final concentration of 1 mM toinduce expression of the acceptor protein and pglB, respectively; afteranother 5 h of incubation, 0.2% w/v L-(+)-arabinose was added again andthe culture left to incubate overnight.

Cells were harvested by centrifugation at 5300×g for 30 min, andpelleted cells were incubated at room temperature for 30 min in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH₂PO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1 percent Tween, 1 mg ml⁻¹ lysozyme and 1 μl ml⁻¹Benzonase nuclease (Novagen). Cell debris was removed by centrifugationat 7840 g for 30 min, the supernatant was collected and 1 ml Ni-NTAagarose (QIAGEN) was added to the supernatant. The slurry-lysate wasincubated for 1 h at 4° C. with shaking then loaded into 10 mlpolypropylene columns (Thermo Scientific). His-tagged ExoA was purifiedby the addition of an elution buffer according to manufacturersinstructions (QIA expressionist, QIAGEN) containing 250 mM imidazolewith the addition of 20 percent glycerol and 5 percent glucose.

Alternatively cells were grown in LB agar plates containingtetracycline, ampicillin, IPTG to a final concentration of 50 μM andL-(+)-arabinose to a final concentration of 0.2% for 16 h at 37° C.Cells were subsequently harvested by scraping and protein purified asindicated above.

Immunoblot Analysis

To verify transfer and presence of the F. tularensis O antigen, sampleswere analysed by western blotting. E. coli cells were grown o/n in 10 mlLB broth and diluted to an O.D.600 nm of 1.0. Cells were centrifuged at13,000 r.p.m. for 10 min, supernatant was removed and cells wereresuspended in 100 μl Laemmli buffer and lysed by boiling for 10 minbefore analysis by western blotting or silver staining. Mouse anti F.tularensis O-antigen monoclonal antibody FB011 (AbCam U.K.) was used ata dilution of 1:1,000, rabbit anti HIS monoclonal antibody was used todetect ExoA at a dilution of 1:10,000 (AbCam U.K.). Secondary antibodiesused were goat anti mouse IRDye680 and IRDye800 conjugates used at1:5000 dilutions. Signal detection was undertaken using the Odyssey®LI-COR detection system (LI_COR Biosciences GmbH).

Cytokine Response Analysis

Spleen supernatants were assessed using mouse inflammatory cytometricbead array kit (CBA-BD biosciences) for IL-10, IL-12p70, IFN-γ, IL-6,TNF-α, and MCP-1. Samples were incubated with the combined capture beadcocktail, and incubated for 1 h at room temperature. Followingincubation, PE detection antibodies were added and incubated for afurther 1 h. Samples were then washed and resuspended in FACS buffer.Cytokine concentrations were measured via quantification of PEfluorescence of samples in reference to a standard curve.

BALB/c Mouse Challenge Studies

Female Balb/C mice were obtained from Charles River Laboratories (Kent,U.K.) at 6-8 weeks of age. The pilot study was done in groups of 10 miceimmunised with either 0.5 μg F. tularensis LPS, 0.5 μg F. tularensisglycoconjugate, 0.5 μg F. tularensis glycoconjugate+SAS, 0.5 μg ‘sham’glycoconjugate+SAS, 0.5 μg ‘sham’ glycoconjugate or SAS only. One groupof mice were left untreated as challenge efficacy controls.Immunisations occurred on days 0, 14 and 28 via intra-peritoneal (IP)route. Mice were challenged 35 days post-immunisation with 100 CFU of F.tularensis strain HN63 by the IP route, delivered in 0.1 ml. Subsequentexperiments used the same schedule with 15 mice per group and doses of10 μg of material per immunisation. Four weeks following finalvaccination 5 mice from each group were tail bled to obtain sera forantibody analysis and culled at day 3 post-infection with spleensharvested to analyse bacterial load and cytokine response. For theenumeration of bacteria, spleen samples were homogenized in 2 ml of PBSthrough 40 μm cell sieves (BD Biosciences) and 100 μl aliquots wereplated onto BCGA plates. F. tularensis LPS-specific IgM and total IgGlevels were determined by ELISA as previously described [19]. All workwas performed under the regulations of the Home Office ScientificProcedures Act (1986).

Statistical Analysis

Statistical analyses were performed using the program PASW (SPSS release18.0). Survival data was analysed by pair-wise Log Rank test stratifiedby experiment. Cytokine and bacterial load data were analysed usingunivariate general linear models, using Bonferroni's post tests tofurther clarify significant differences.

Production and Purification of Glycoconjugate Vaccine

E. coli CLM24 carrying the vectors pGAB2, pGVXN114 and pGVXN150 wasgrown for 16 h in 200 mL LB broth at 37° C., 180 rpm. This was used toinoculate 1.8 L of LB broth and further incubated at 110 rpm. 37° C.until an O.D600 reading of 0.4 to 0.6 was reached. At this pointL-(+)-arabinose was added to a final concentration of 0.2% and IPTG to afinal concentration of 1 mM to induce expression of exoA and CjpglBrespectively; after another 5 hours of incubation, 0.2% w/vL-(+)-arabinose was added again and the culture left to incubate o/n.

Cells were harvested by centrifugation at 6,000 rpm. for 30 m, andpelleted cells were incubated at room temperature for 30 m in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1% Tween, 1 mg/ml lysozyme and 1 μl/ml Benzonasenuclease (Novagen). Cell debris was removed by centrifugation at 10,000r.p.m. for 30 m, the supernatant was collected and 1 ml Ni-NTA agarose(QIAgen) was added to the supernatant. The slurry-lysate was incubatedfor 1 h at 4° C. with shaking then loaded into 10 ml polypropylenecolumns (Thermo scientific). His tagged ExoA was purified by theaddition of an elution buffer according to manufacturer's instructions(QIA expressionist, QIAGEN) containing 250 mM imidazole with theaddition of 20% w/v glycerol and 5% w/v glucose. Protein yields wereestimated using a bicinchonic acid assay kit according to manufacturer'sinstructions (Pierce® Biotechnology BCA protein Assay Kit, U.S.A.).

For large-scale protein purification, material was isolated using GEHealthcare HIS trap columns and an AKTA purifier with an imidazolegradient of 30 mM to 500 mM. The collected fraction containing ExoAglycosylated with F. tularensis O-antigen was further purified using aresource Q anionic exchange column (GE Healthcare) with a NaCl gradientfrom 0 to 500 mM in 20 mM TrisHCl pH 8.0. This generated a typical yieldof 2-3 mg/ml of glycoconjugate per 2 L of E. coli culture.

The same techniques were used for the generation of the ‘sham’ C. jejuniheptasaccharide ExoA glycoconjugate. The plasmid coding for thisheptasaccharide was pACYCpgi carrying the entire CAI cluster from C.jejuni 81116 [1].

Protein Expression

Escherichia coli strain CLM24 with a chromosomally inserted copy of pglBwere grown in Luria-Bertani (LB) broth at 37° C., with shaking.Antibiotics were used at the following concentrations: tetracycline 20μg ml-1 and ampicillin 100 μg ml-1. Tetracycline was used to maintainthe plasmid pGAB2 coding for Francisella O-antigen and ampicillin wasused to maintain the plasmid coding for the acceptor carrier protein.Additionally, a final concentration of 4 mM Manganese chloride was addedas an additional co-factor to the cultures.

E. coli cells were grown for 16 h in 200 ml LB broth at 37° C., withshaking. This was used to inoculate 1.8 l of LB broth and furtherincubated with shaking at 37° C. until an OD600 reading of 0.4-0.6 wasreached. At this point L-(+)-arabinose was added to a finalconcentration of 0.2% w/v, 4 mM final concentration of manganesechloride, and IPTG to a final concentration of 1 mM to induce expressionof the acceptor protein and pglB, respectively; after another 5 h ofincubation, 0.2% w/v L-(+)-arabinose was added again and the cultureleft to incubate overnight.

Cells were harvested by centrifugation at 5300×g for 30 min, andpelleted cells were incubated at room temperature for 30 min in a lysissolution composed of 10× BugBuster protein extraction reagent (Novagen)diluted to 1× in 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0supplemented with 0.1 percent Tween, 1 mg ml-1 lysozyme and 1 μl ml-1Benzonase nuclease (Novagen). Cell debris was removed by centrifugationat 7840 g for 30 min, the supernatant was collected and 1 ml Ni-NTAagarose (QIAGEN) was added to the supernatant. The slurry-lysate wasincubated for 1 h at 4° C. with shaking then loaded into 10 mlpolypropylene columns (Thermo Scientific). His-tagged ExoA was purifiedby the addition of an elution buffer according to manufacturersinstructions (QIA expressionist, QIAGEN) containing 250 mM imidazolewith the addition of 20 percent glycerol and 5 percent glucose.

Creation of WecA⁻ E. coli Strain

The kanamycin resistance cassette from plasmid pKD4 was amplified usingthe following primers KnF5′-GTGAATTTACTGACAGTGAGTACTGATCTCATCAGTATTTTTTTATTCACTGTGTAGGCTGGAGCTGCTTC-3′ (SEQ ID NO 28) and KnR5′-GTAAAACGCAGACTGCGTAGAAATCGTGGTGGCAGCCCCAATTTAACCAACATATGAATATCCTCCTTAGCTGCAG-3′ (SEQ ID NO 12) using accuprime taq hifi(Invitrogen UK) and the following cycling conditions 94° C./30 s,followed by 30 cycles of 94° C./30 s, 56° C./30 s, 68° C./90 s. Theseprimers carry 5′ end tails that are homologous to the wecA gene ofEscherichia coli K-12. The PCR product was digested with Dpnl andpurified before transforming 1 μg into E. coli K 12 carrying, the lambdared helper plasmid pKD20 (Datsenko and Wanner PNAS, One-stepinactivation of chromosomal genes in Escherichia coli K-12 using PCRproducts, Jun. 6, 2000 pp. 6640-6645). Bacteria were grown on LB agarplates containing 10 mM L-arabinose to induce rec recombinase expressionfor 48 hrs prior to plating on kanamycin plates. wecA gene deletionswere detected by gene specific PCR and resistance to kanamycin.

Animals

Animals were kept in accordance with the Animals (Scientific Procedures)Act 1986. Codes of Practice for the Housing and Care of Animals used inScientific Procedures 1989. Following challenge with F. tularensis, allanimals were handled under UK Advisory Committee on Dangerous Pathogensanimal containment level 3 conditions within a half-suit isolatorcompliant with British Standard BS5726.

Female Fischer 344 rats were obtained from Harlan, UK. Rats wereimplanted with biotherm microchips sub-cutaneously.

Rats were vaccinated with LVS in PBS via the sub-cutaneous (s.c.) route.Rats were vaccinated with 10 μg glycoconjugate in 100 μl PBS via thes.c. or intra-peritoneal route 3 times, 2 weeks apart. Aerosol challengewith F. tularensis Schu S4 occurred five weeks following finalvaccination.

Following challenge, animals were observed twice daily, and signs ofdisease and sub-cutaneous temperature recorded. Disease signs wereassigned a score, and a cumulative score for disease observed wascalculated. Animals were weighed once daily. Weight data was analysedusing IBM SPSS V21.0. Weight data was taken as the differential weightfrom 1 day prior to challenge. Data was found to fit the normal,Gaussian distribution using Q-Q plots (not shown). The data was thenanalysed using a repeated measures General Linear Model. Validity of thedata for this test was further established using Levene's tests forunequal variance (not shown). Individual comparisons, pairwise anddependant or independent of time points, were performed using theBonferroni's correction. Humane end points of more than 15% weight loss,and/or sub-cutaneous temperature reading of less than 33° C. were used.Animals underwent Schedule 1 euthanasia with i.p. administered euthatal.

Aerosol Challenge

Rats were exposed to an aerosol of F. tularensis SchuS4 by theinhalational route in a nose-only exposure unit (Dstl, in house)utilising a 6-jet Collison atomiser (Dstl, in house) attached to acontained Henderson Piccolo arrangement to condition the aerosol to 50%(±5%) relative humidity, and controlled by the (AeroMP) AerosolManagement Platform aerosol system (Biaera Technologies L.L.C.). Theanimals were exposed to the aerosolised bacteria for 10 minutes, withimpingement of the aerosol cloud sampled at the midway point ofchallenge into PBS via an All-Glass Impinger (AGI-30; Ace Glass,Vineland, N.J.).

Following challenge, the impinged aerosol was enumerated by serialdilution and plating onto BCGA plates. Calculated retained dose wascalculated from aerosol concentration (cfu/L of air), using Guyton'sformula (27), for minute respiratory volume, and assuming 40% retentionof 1-3 μm droplets (26).

Cell Isolation and Culture

Rat spleens were homogenised through a 40 μm sieve using a sterileplunger and colleved into L15 medium. The isolated splenocytes werediluted to 2×10⁶ cells/ml in medium and cultured in the presence ofeither medium alone, sonicated LVS whole cells (10 μg/ml, Dstl),sonicated Schu S4 whole cells (10 μg/ml, Dstl), purified ExoA (5 μg/ml,LSHTM) or Concanavalin-A (Con-A, 5 μg/ml, Sigma-Aldrich). For culturesof cells from LVS infected or PBS control rats, splenocytes were dilutedin L-15 medium (Life Technologies) supplemented with 10% Foetal BovineSerum (Sigma), non-essential amino acids (Life Technologies),2-mercaptoethanol (Life Technologies), 100 U/ml penicillin and 100 mg/mlstreptomycin sulphate (Life Technologies) and then cultured at 37° C. inthe absence of a controlled CO₂ environment. For cultures of cells fromExoA vaccinated rats, splenocytes were diluted in RPM11640 medium (LifeTechnologies), supplemented as described above and then cultured at 37°C. with 5% CO₂.

Measurement of IFNγ by Enzyme Linked Immunosorbent Assay (ELISA).

Splenocytes (2×10⁵ per assay well) were cultured in duplicate in thepresence of antigen for 72 hours and supernatants harvested and storedat −20° C. prior to use. The expression of IFNγ was determined in plasmasupernatants using a commercial rat IFNγ ELISA kit (Mabtech) withresponses determined by measurement of optical density at 450 nm(OD_(450 nm)). For reporting, the OD_(450 nm) results were normalised bytransformation into units of ρg/ml by generating a standard curve usingrecombinant rat IFNγ, as supplied with the assay kit.

Flow Cytometry.

Splenocytes (2×10⁶ cells per assay well) were cultured in the presenceof antigen for 20 hours with Brefeldin-A (10 ug/ml, Sigma-Aldrich) addedto culture medium for the final 4 hours of the culture. Cells wereharvested by centrifugation (300 g/5 mins) and stained using thefollowing anti-rat surface marker antibodies; CD3-Brilliant Violet 421,CD4-FITC (clone OX35, eBioscience) and CD8-PerCP eFluor710 (clone OX8,eBioscience). Cells were stained for 15 minutes at 4° C. in the presenceof a fixable yellow (405 nm) cell viability dye (Life Technologies) andthen fixed for 16 hours at 4° C. in Cytofix fixation reagent (BDBiosciences). Fixed cells were permeabilised in BD BiosciencesPermeabilistation Buffer and then stained intracellularly for 30 minutesat 4° C. using anti-rat antibodies IFNγ-PE (clone DB-1, BD Biosciences)and IL17A-APC (clone eBio17B7, eBioscience). Stained samples wereanalysed using a FACSCanto II analyser equipped with 405, 488 and 633 nmlasers (BD Biosciences). An example gating strategy for measurement ofintracellular expression of IFNγ and IL-17 is given in FIG. 25. Dataanalysis was performed using FlowJo v10 software (TreeStar, USA). Allantibodies were titrated prior to use to ensure optimal staining. Mediannumber of live-singlet lymphocyte cell events on which analyses wereperformed was 62,598 (±18,724 SD)

ImageStream Staining and Data Capture

500 μl whole rat blood was blocked with 5 μl anti-rat CD32 antibody (BDPharmingen Cat No: 550271), incubated for 5 minutes at room temperature(“23° C.). Following blocking 40 μl anti-rat CD45-FITC (BD PharmingenCat No: 554877), 50 μl anti-rat CD3-APC (BD Pharmingen, Cat No: 557030)and 120 μl 0.5% BSA in PBS were added to the blood sample. Samples werethen incubated at 4° C. for 45 minutes. Blood samples were subsequentlycentrifuges at 300× g for 10 minutes and the supernatants removed. 1 mlBD lysis buffer (BD Pharmingen) was added and samples incubated for 10minutes at room temperature (“23° C.), before being centrifuged at 300×gfor 10 minutes. Supernatants were removed and samples were washed andcentrifuged again before being re-suspended in 100 μl of 4%paraformaldehyde and stored at 4° C. Samples were counterstained with 3μM 4′,6-diamidino-2-phenylindole (DAPI) 3 minutes before data capture.

Data was collected using an dual camera imagestream X Mk II equipt witha 405 nm laser (set to 2 mW), a 488 nm laser (set to 100 mW) and a 642nm laser (set to 150 mW). Samples were acquired using Inspire (version2.0) where a minimum of 20,000 in-focus (Gradient RMS <50) nucleated(Intensity Ch07<1×10⁴) were captured per rat. Single stained sampleswere also created and acquired for the construction of a compensationmatrix. This was performed in inspire using the compensation wizardduring data capture. Data are generated from a minimum of 4 mice.Statistical analysis was completed using GraphPad Prism version 6.02.Data validity was first assessed using a Brown-Forsythe test forvariance after which a two-way ANOVA was completed with Dunnett'smultiple comparisons test was completed.

ELISA for Anti Gt-ExoA Antibody Titre

Plates were coated with 5 μg/ml Gt-ExoA in PBS and, 100 μl per well, andincubated at 4° C. overnight. After blocking with 1% skimmed milk powderin PBS for 2 hours at 37° C., plates were washed three times with 0.05%TWEEN in PBS. Sera was applied to plates at 1:50 and serially diluted1:2 across the plate, in 1% skimmed milk powder. Bound IgG rat antibodywas detected using anti-rat antibody conjugated to HRP at 1:2000 in PBS,and developed using 10 mM2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) in citratebuffer, with 0.01% H₂O₂. OD was measured at 414 nm. Antibody titre wasdefined as the reciprocal of the highest dilution of serum that had amean OD value at least 3 standard deviations higher than the mean OD ofnon-vaccinated serum.

EXAMPLE 1

Fischer 344 Rats are Susceptible to <10 CFU F. tularensis Schu S4 Viathe Aerosol Route

Groups of 5 rats were challenged with a range of doses of Francisellatularensis Schu S4 via the aerosol route, from approximately 9 cfu to3.15×10⁴ cfu. To determine bacterial dissemination following challenge,groups of five rats from each dose group were sacrificed at 7 days postinfection. At this time, all rats challenged with 3.15×10⁴ cfu, and 3 of5 rats challenged with 2.22×10³ had already succumbed to infection. At 7days post-infection, all infected animals had highly colonised lungs,containing greater than 1×10⁶ cfu/g of lung tissue. The most heavilycolonised lungs, from animals challenged with 2.22×10³ contained up to1×10⁸ cfu/g. Bacteria had disseminated from lungs to liver and spleen inall infected rats. All rat spleens and livers at 7 days post infectioncontained greater than 1×10⁴ cfu/g tissue Schu S4 bacteria.

All rats challenged with 2.94×10² to 3.15×10⁴ cfu rats succumbed toinfection within 14 days of challenge (FIG. 19A). Of rats challengedwith CFU of F. tularensis, only 1 animal of 5 survived to the end ofexperiment at 21 days post infection. During the recovery of thisanimal, its disease signs resolved and some weight was recovered. TheMLD is therefore estimated to be less than 9 cfu via the aerosol route.All infected rats underwent a febrile stage, with subcutaneoustemperatures which were raised at least 1.5° C. above their baselinetemperature (data not shown). All infected rats showed severe signs ofdisease. Rats first became ruffled, and developed eye problems includingsecretion of porphyrin and ptosis of the eyelids until their eyes werecompletely closed, followed by hunched posture alongside rapidbreathing. Rats became more lethargic and less responsive to stimuliover the course of disease (FIG. 19B). All infected rats lost weight, ina dose dependent manner (FIG. 19C). Rats which received the highestchallenge: 3.15×10⁴ cfu, rapidly lost between 7 and 10 percent of theirbody weight within 5 days of challenge. Those rats which received alower challenge all lost at least 10 percent of their starting weight,and in some cases more than 25 percent of body weight, but over agreater length of time.

EXAMPLE 2

LVS Disseminates to Lungs, Liver, and Spleen Following Challenge,Inducing Expansion of Neutrophil and Macrophage Populations in theBlood.

To determine dissemination and clearance of bacteria, and determine hostcell responses to LVS infection, groups of five rats were vaccinatedwith 5.56×10⁵ or 5.56×10⁷ CFU of LVS sub-cutaneously. Followingvaccination, rats were sacrificed at 3, 7 and 21 days post-vaccinationto determine bacterial dissemination and clearance. There was nosignificant difference in colonisation of organs between rats vaccinatedat the 2 different doses. At 3 days post-vaccination, all rats werecolonised in the liver and spleen, whilst lungs had low numbers ofdetectable bacteria. At 7 days, no bacteria could be detected in lungsand liver of vaccinated rats, whilst spleens were still colonised. Nobacteria were detected in lungs, liver or spleen at 21 days postinfection.

Rat immune cells in whole blood were quantified using the ImageStream XMkll. Cell types were differentiated by CD45 intensity verses sidescatter intensity identifying three distinct populations namely,lymphocytes, monocytes/macrophages and neutrophils (FIGS. 20A and B).There was a significant decrease in lymphocyte counts in rats challengedwith both 10⁵ and 10⁷ LVS at day 3 post vaccination (p<0.01), with areturn to baseline at day 7 and day 21. This is mirrored in theneutrophil count, with a significant increase in neutrophils at day 3 inboth dose groups decreasing back to baseline at day 7 and 21.Monocyte/macrophage numbers in rats challenged with 10⁷ LVS weresignificantly increased at day 3, returning to baseline levels at days 7and 21.

EXAMPLE 3

LVS Induces T Cell Memory Response Characterised by IFNγ and IL-17Expression on Re-Stimulation

To determine the splenocyte re-stimulation response following LVSvaccination, 5 rats were sacrificed at day 21 and day 35post-vaccination and splenocytes were harvested from rat spleens. IFNγsecretion by splenocytes was measured following re-stimulation with arange of antigens. The absence of responses in the PBS control ratsindicates the antigen specific nature of these memory re-call responses,whilst all cells secreted IFNγ in response to the mitogen ConAindicating the cells potential for responding. LVS vaccinated ratsplenocytes secreted significantly more IFNγ in response to stimulationwith all three F. tularensis whole cell antigen preparations than PBSvaccinated rats (FIG. 21).

FIG. 4. Antigen Stimulated IFNγ Response in LVS Infected Rats.

Splenocytes isolated from rats 21 days following infection with 10⁵ CFULVS (grey bars), 10⁷ CFU LVS (black bars) or the PBS controls (hashedbars) were cultured in the presence of LVS sonicate, F. tularensissonicate, Con-A or medium and then expression of IFNγ in 72 hour culturesupernatants measured by ELISA. IFNγ responses (ρg/ml) are presented asmean response for each group (n=5)±SEM.

Further investigation of memory response to LVS was investigated bymeasurement of intracellular expression of IFNγ and IL-17 followingantigen stimulation, 21 days post-infection with LVS. LVS infectionresulted in the induction of both CD4 and CD8 effector memory responses,as demonstrated by antigen-specific IFNγ responses by these cellphenotypes (FIG. 22). Additionally, CD4+ memory cells that express IL-17upon antigen stimulation are also generated by LVS infection in the rats(FIG. 22).

EXAMPLE 4

Glycoconjugate Induces Antigen Specific Memory Response and IgG Antibody

Similarly to LVS vaccinated rats, splenocytes were isolated fromglycoconjugate vaccinated rats at 35 days post final vaccination. Cellswere re-stimulated both with crude antigen preparations, and recombinantExoA protein for 72 hours. IFNγ secretion following re-stimulation ofsplenocytes from LVS challenged rats with crude F. tularensis antigens,corroborates intracellular IFNγ expression previously described (FIG.23). In addition, the measurement of ExoA antigen specific IFNγresponses only in Gt-ExoA vaccinated rats confirmed the recognition ofthe Gt-ExoA conjugate by the cell mediated compartment of the immunesystem (FIG. 23). Serum from Gt-ExoA vaccinated rats and theirappropriate MF59 only controls were measured for IgG recognition of GtExoA. Sera from MF59 only controls showed no appreciable binding toGt-ExoA. IgG titres from rats vaccinated with Gt-ExoA by the i.p. routewere 1:204800, and by the s.c. route 1:102400, demonstrating an antigenspecific IgG response to vaccine.

EXAMPLE 5

LVS and F. tularensis O-Antigen+Exoprotein a Glycoconjugate Both ProtectAgainst Pulmonary Tularemia

Rats were vaccinated with 5.39×10⁷ LVS via the s.c. route, andglyconojugate by both the s.c. and i.p. route. Five weeks later theanimals were challenged with an average 5.48×10² F. tularensis Schu S4.All vaccinated rats survived 21 days post aerosol challenge (FIG. 24A).Of five rats vaccinated with MF59 alone via the i.p route, one ratsurvived to 21 days post infection (FIG. 24A). Three of five ratsvaccinated with MF59 alone via the s.c. route survived to 21 days postinfection, which is not significantly different from rats vaccinatedwith glycoconjugate via the s.c. route (FIG. 24A).

Rats vaccinated with Gt-ExoA by either route, or with LVS did not becomefebrile (data not shown) and showed no signs of disease (FIG. 24B).Comparatively, rats vaccinated with PBS s.c. and adjuvant only via i.pand s.c. routes all become febrile (data not shown), and all showedsevere signs of disease (FIG. 24B)

Weight change in rats after challenge, compared to 1 day beforechallenge, was shown to be significantly different over time between allgroups of vaccinated rats and their relevant controls (FIG. 24C). Weightchange between rats vaccinated with Gt-ExoA by the s.c. route and therelevant control rats, vaccinated with MF59 alone by the s.c. route,significantly diverged on day 2 after challenge FIG. 24C.2) (p<0.05).Weight change in rats vaccinated with LVS or with Gt-ExoA via the i.p.route significantly diverged from their apposite controls on day 4(FIGS. 24C.1 and 24C.3) (p<0.005, p<0.0005 respectively).

The surviving control rats all resolved signs of disease and hadrecovered some weight by 21 days following infection. F. tularensis wasnot detected in lungs, liver or spleen of LVS and glycoconjugatevaccinated rats at 21 days post infection whilst all surviving MF59 onlyvaccinated rats were colonised with F. tularensis in lung, liver andspleen at 21 days post infection.

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1. A vaccine or immunogenic composition comprising a carrierpolypeptide, wherein the carrier polypeptide comprises: one or moreT-cell dependent epitopes; one or more amino acid sequences having atleast one amino acid motif comprising the amino acid sequenceD/E-X-N-X-S/T, wherein X is any amino acid except proline; and anO-antigen antigenic polysaccharide isolated from Francisella crosslinkedto said carrier polypeptide.
 2. The vaccine or immunogenic compositionaccording to claim 1 wherein said O-antigen comprises:2-acetamido-2-deoxy-O-D-galact-uronamide, 4,6-dideoxy-4-formamido-D-glucose and/or 2-acetamido-2,6-dideoxy-O-D-glucose and/or N-acetyl glucosamine, or comprises orconsists of4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNAcAN is 2-acetamido-2-deoxy-O-D-galacturonamide, Qui4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose, or comprises or consist of4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-GlcNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNacAN is 2-acetomido-2-deoxy-O-D-galacturonamide, Qui4NFm is4, 6-dideoxy-4-formamido-D-glucose and the reducing end group GlcNAc isN-acetyl glucosamine.
 3. The vaccine or immunogenic compositionaccording to claim 1, wherein said O-antigen polysaccharide is atetrasaccharide.
 4. The vaccine or immunogenic composition of claim 1,wherein said carrier polypeptide, comprises the amino acid sequence ofSEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, or SEQ ID NO: 7, comprises a polymorphic sequence variantof the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, comprises apolypeptide fragment of the amino acid sequence of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ IDNO: 7, or comprises one or more glycosylation motifs of the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
 7. 5.-10. (canceled)
 11. Thevaccine or immunogenic composition according to claim 1, wherein saidcomposition includes an adjuvant.
 12. The vaccine or immunogeniccomposition according to claim 11 wherein said adjuvant is an aluminiumbased adjuvant suitable for use in a human subject.
 13. A method oftreating or preventing a Francisella infection comprising: administeringthe vaccine or immunogenic composition of claim 1 to a subject, therebytreating or preventing the Francisella infection in the subject.
 14. Anantigenic polypeptide comprising: a carrier polypeptide comprising oneor more T-cell dependent epitopes and one or more amino acid sequenceshaving at least one amino acid motif comprising the amino acid sequenceD/E-X-N-X-S/T wherein X is any amino acid except proline and crosslinkedto said carrier polypeptide is an antigenic polysaccharide wherein thepolysaccharide is isolated from Francisella and is an O-antigen.
 15. Theantigenic polypeptide according to claim 14 wherein said carrierpolypeptide, comprises the amino acid sequence as set forth in of SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ IDNO: 6, or SEQ ID NO: 7, comprises a polymorphic sequence variant of theamino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, comprises apolypeptide fragment of the amino acid sequence of SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ IDNO: 7, or comprises one or more glycosylation motifs of the amino acidsequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
 7. 16.-21. (canceled)
 22. Theantigenic polypeptide according to claim 14, wherein said O-antigencomprises: 2-acetamido-2-deoxy-O-D-galact-uronamide, 4,6-dideoxy-4-formamido-D-glucose and/or 2-acetamido-2,6-dideoxy-O-D-glucose and/or N-acetyl glucosamine, or comprises orconsist of:4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-QuiNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNAcAN is 2-acetamido-2-deoxy-O-D-galacturonamide, Qui4NFm is4,6-dideoxy-4-formamido-D-glucose and the reducing end group QuiNAc is2-acetamido-2,6-dideoxy-O-D-glucose, or comprises or consists of:4)-α-D-GalNAcAN-(1-4)-α-D-GalNAcAN-(1-3)-β-D-GlcNAc-(1-2)-β-D-Qui4NFm-(1-),wherein GalNacAN is 2-acetomido-2-deoxy-O-D-galacturonamide, Qui4NFm is4, 6-dideoxy-4-formamido-D-glucose and the reducing end group GlcNAc isN-acetyl glucosamine.
 23. A bacterial cell genetically modified toinclude: i) a nucleic acid molecule comprising the nucleotide sequenceof the Francisella O-antigen biosynthetic polysaccharide locus [SEQ IDNO: 8]; ii) a nucleic acid molecule comprising a nucleotide sequence ofan oligosaccharyltransferase; and/or iii) a nucleic acid moleculecomprising a nucleotide sequence of carrier polypeptide wherein the acarrier polypeptide comprising one or more T-cell dependent epitopes andone or more amino acid sequences having at least one amino acid motifcomprising the amino acid sequence D/E-X-N-X-S/T wherein X is any aminoacid except proline, wherein said bacterial cell is adapted forexpression of each nucleic acid molecule and synthesizes an antigenicpolypeptide according to claim
 14. 24. The bacterial cell according toclaim 23 wherein said nucleic acid molecule encoding anoligosaccharyltransferase comprises the nucleotide sequence of SEQ IDNO: 9, 23, 25 or 26 or a polymorphic sequence variant thereof, whereinsaid variant comprises a nucleic acid molecule the complementary strandof which hybridizes under stringent hybridization conditions to thesequence of SEQ ID NO: 9, 23, 25 or 26 and wherein said nucleic acidmolecule encodes an oligosaccharyltransferase;
 25. The bacterial cellaccording to claim 23, wherein said carrier polypeptide is encoded by anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:10, 11, 13, 14, 15, 16 or 29, or a nucleic acid molecule thecomplementary strand of which hybridizes under stringent hybridizationconditions to the sequence of SEQ ID NO: 10, 11, 13, 14, 15, 16 or 29.26. The bacterial cell according to claim 23, wherein said bacterialcell expresses a glycosyltransferase encoded by a nucleic acid moleculecomprising the nucleic acid sequence of SEQ ID NO 17, or a sequencevariant thereof wherein said variant comprises a nucleic acid moleculethe complementary strand of which hybridizes under stringenthybridization conditions to the sequence set forth in SEQ ID NO: 17 andwherein said nucleic acid molecule encodes an glycosyltransferase. 27.The bacterial cell according to claim 23, wherein saidglycosyltransferase encoded by a nucleic acid molecule comprising thenucleic acid sequence of SEQ ID NO 17 is modified wherein saidmodification is the addition, substitution or deletion of at least onenucleic acid base wherein said modified nucleic acid sequence encodes aglycosyltransferase polypeptide which has reduced or undetectable enzymeactivity when compared to an unmodified glycosyltransferase polypeptide.28. The bacterial cell according to claim 27 wherein said cell isdeleted for all or part of the nucleic acid sequence of SEQ ID NO: 17 toprovide a non-functional glycosyltransferase polypeptide.
 29. Thebacterial cell according to claim 23, wherein at least theoligosaccharyltransferase is integrated into the bacterial genome toprovide a stably transfected and expressing oligosaccharyltransferase.30. The bacterial cell according to claim 23, wherein one or morenucleic acid molecules encoding carrier polypeptides are also integratedinto the bacterial genome.
 31. A bacterial cell culture comprising agenetically modified bacterial cell according to claim
 23. 32. A processfor producing one or more glycoconjugates, comprising: i) providing thebacterial cell culture according to claim 31; ii) providing cell cultureconditions; and iii) isolating one or more glyconjugates from thebacterial cell or cell culture medium.
 33. A cell culture vesselcomprising the bacterial cell culture according to claim
 31. 34. Thecell culture vessel according to claim 33, wherein said cell culturevessel is a fermentor.